Over the past decade antibodies imposed themselves as one of the most promising therapeutic approaches, in particular in the field of oncology, as well as an important source of research or diagnosis tools.
The Immunoglobulin G (IgG) is the basic structure of a typical antibody, comprising two heterodimers of heavy and light chains bond together by disulphide bridge. Natural single chain antibodies have however been discovered in at least two groups of animals: Camelidae (Hamers-Casterman et al, 1993, Nature, 363, pp446-448) and sharks (Greenberg et al, Nature. 1995, Mar. 9; 374(6518):168-73). These single chain antibodies constitute an additional class of IgG devoid of light chain. The recognition part of these single chain natural antibodies includes only the variable domain of the heavy chain called VHH. VHHs contain four frameworks (FR) that form the scaffold of the IgG domain and three complementarity-determining regions (CDRs) that are involved in antigen binding.
Many advantages of VHHs scaffold have been reported: without interchain disulfide bridges, they are generally more soluble and stable in a reducing environment (Wesolowski et al, 2009 Med Microbiol Immunol. August; 198(3):157-74). VHH have also been reported to have higher solubility, expression yield and thermostability due to their small size (15 kDa) (Jobling S A et al, Nat Biotechnol. 2003 January; 21 (1): 77-80). Moreover, VHH frameworks show a high sequence and structural homology with human VH domains of familly III (Muyldermans et al, 2001. J Biotechnol. June; 74 (4): 277-302) and VHHs have comparable immunogenicity as human VH and thus constitute very interesting agents for therapeutic applications, some of them are currently in phase II clinical trials (Ablynx Nanobodies®). On ten amino acids differing from human, four hallmark aminoacids of VHH have been identified in the framework-2 region.
The properties of VHH scaffolds have many advantages, for use in therapy: they have a better penetration in tissues, a faster clearance in kidneys, a high specificity but also reduced immunogenicity.
Camelid antibody libraries have been described for example in US2006/0246058 (National Research Council of Canada). The described phage display library comprises fragments of llama antibodies, and especially single domain fragments of variable heavy chains (VHH and VH). The libraries were made using lymphocyte genomes of non-immunized animals (naïve library). The resulting phage display library also contains contaminants of conventional VH antibody fragments.
U.S. Pat. No. 7,371,849 (Institute For Antibodies Co., Ltd) also reports methods of making VHH library from VHH genes of camelids. The diversity of such library was obtained by improving the conventional process of isolating VHH variable regions from naïve repertoire.
However, these prior art do not address the issue of immunogenicity from non-human derived antibodies. Even if some of them are identified to bind specific target of interest, they can not be administered in patients for use as therapeutics without the risk of activating the human immune system.
A method to humanize a camelid single-domain antibody is described in Vincke et al, 2008, JBC Vol 284(5) pp 3273-3284.
U.S. Pat. No. 8,367,586 discloses a collection of synthetic antibodies or their fragments. These antibodies comprise variable heavy chain and variable light chain pairs and have, in their framework region, a part of optimal germline gene sequences. This incorporation of human sequence allows to decrease the risk of immunogenicity for therapeutic use.
Monegal et al (2012, Dev Comp Immunol. 36(1):150-6) reports that single domain antibodies with VH hallmarks are repeatedly identified during biopanning of llama naïve libraries. In fact, VH hallmarks are more frequently identified on the binders selected from VHH naïve library, than VHH hallmarks. For example, Monegal et al have shown that 5% of VH hallmarks are found in the naïve library, while 20% of these VH hallmarks are found among the antibodies selected following biopanning against antigens.
Therefore, despite this knowledge, there is still a need to provide single domain antibody libraries, with high diversity, and capable of generating highly stable, humanized single domain antibody with high affinity with a desired target.
Accordingly, one aspect of the disclosure is to provide a non-immune, recombinant single domain antibody library, of high diversity, capable of generating highly stable single domain antibody library with high affinity against specific antigen. Another aspect is to provide a library enriched in single domain antibodies active in the intracellular environment. Yet another aspect is to provide a library enriched in single domain antibodies with high thermostability.